Labeling is a tool for marking a protein, cell, or organism of interest and plays a prominent role in many biochemistry, molecular biology and medical diagnostic applications. A variety of different labels have been developed, including radiolabels, chromolabels, fluorescent labels, chemiluminescent labels, etc.
Fluorescent labels that are increasing in application are fluorescent proteins. Various fluorescent proteins have been described. For example, green fluorescent protein (GFP), a protein of the jellyfish Aequorea victoria, has an excitation maximum at 395 nm, a second excitation peak at 475 nm and an emission maximum at 510 nm. Other fluorescent proteins derived from Anthozoa species, e.g., corals, have been described. See the Literature section, below.
Fluorescent proteins are used in a wide variety of applications involving labeling of a protein, a cell, or a subcellular structure. Such applications include assessing gene expression during development of a multicellular organism, during the process of cellular differentiation, in response to a drug or other inducer of promoter activity. In these types of applications, a fluorescent protein is frequently used as a reporter to serve as a read-out of promoter activity. Other applications include monitoring intracellular protein movement or translocation, e.g., from one subcellular compartment to another, and monitoring protein intercellular protein movement.
Currently available fluorescent proteins exhibit emission spectra that do not change over time. Certain studies cannot be conducted effectively using these fluorescent proteins. For example, one cannot accurately study transient protein expression using fluorescent proteins currently available because one cannot tell from the signal of the protein whether the protein is newly synthesized or has been present in the cell for a long period of time.
As such, there is great interest in developing fluorescent proteins that change emission spectra over time.
Literature
For GFP, see, e.g., Haas, et al. (1996) Current Biology 6:315–324; Yang, et al. (1996) Nucleic Acids Research 24:4592–4593. GFP crystal structure is reported in Ormö et al. (1996) Science 273:1392–1395; and Yang et al. (1996) Nature Biotechnol 14:1246–1251. For Anthozoa-derived fluorescent proteins, see, e.g., WO 00/34318, WO 00/34319, WO 00/34320, WO 00/34321, WO 00/34322, WO 00/34323, WO 00/34324, WO 00/34325, WO 00/34326, and WO 00/34526. See also Matz et al. (1999) Nature Biotechnol. 17:969–973; and Terskikh et al. (November, 2000) Science 290:1585–1588.